title: Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.
abstract: Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.
authors.affiliation: Laboratoire de Parasitologie Médicale, Biotechnologies et Biomolécules, Institut Pasteur de Tunis, LR11-IPT06, 13 Place Pasteur - BP74, 1002 Tunis-Belvédère, Tunisia.
authors.forename: Imène
authors.initials: I
authors.lastname: Turki
authors.forename: Akil
authors.initials: A
authors.lastname: Hammami
authors.forename: Habib
authors.initials: H
authors.lastname: Kharmachi
authors.forename: Mohamed
authors.initials: M
authors.lastname: Mousli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000276:immunology / Q000627:therapeutic use*; D057134:Antibodies, Neutralizing / Q000276:immunology; D000914:Antibodies, Viral / Q000276:immunology; D000915:Antibody Affinity / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D017122:Bacteriophage T4 / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D006023:Glycoproteins / Q000276:immunology*; D007116:Immunization, Passive; D009500:Neutralization Tests; D011818:Rabies / Q000276:immunology / Q000517:prevention & control / Q000628:therapy*; D011819:Rabies Vaccines / Q000276:immunology; D011820:Rabies virus / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D057127:Single-Chain Antibodies / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use*; D014759:Viral Envelope Proteins / Q000276:immunology*
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D000956:Antigens, Viral; D006023:Glycoproteins; D011819:Rabies Vaccines; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D014759:Viral Envelope Proteins; C064951:glycoprotein G, Rabies virus
keywords: Antibody engineering; ELISA; Fd; Foldon; Neutralization; RFFIT; RIG; RV; RVG; Rabies virus; Trimeric scFv; enzyme-linked immunosorbent assay; foldon motif; mAb; monoclonal antibody; rabies immunoglobulin; rabies virus; rabies virus glycoprotein; rapid fluorescent focus inhibition test.; scFv; single-chain variable fragment